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A complex of interacting DNAase I-hypersensitive sites near the drosophila glue protein gene, Sgs4
Cell, Vol. 29, 601-607, June 1962, Copyright 0
1962
by MIT
A Complex of Interacting Sites near the Drosophila Antony W. Shermoen and Steven Department of Molecular Biology University of California Berkeley, California 94720
K. Beckendorf
Summary The chromatin structure adjacent to the Drosophila glue protein gene Sgs4 changes drastically when the gene is active. In nuclei from embryos or tissue culture cells in which Sgs4 is inactive, there are three DNAase l-hypersensitive sites 3’ to the gene, but none near its 5’ end. In the nuclei of late third instar salivary glands, Sgs4 is actively transcribed, and a complex of five DNAase l-hypersensitive sites appears 5’ to the gene. Two of the sites are near the point of transcription initiation, at -70 and +30. The others are much farther from the gene at -330, -405 and -480 and are affected by small deletions: one deletion reduces expression about 50fold and removes sequences corresponding to the -330 hypersensitive site; another makes no Sgs4 RNA and removes sequences corresponding to two hypersensitive sites, -405 and -480. Thus the hypersensitive sites, or DNA sequences within 50 bp of them, seem to be required for normal gene expression. Distinct changes are seen in the chromatin from salivary glands of these mutant strains. The first strain is simply missing the -330 hypersensitive site, while the second is missing all of the tissue-specific 5’ sites, even though sequences corresponding to three of them remain. This suggests that hierarchical interactions among the regions 5’ to Sgs4 are required for its full expression. A sequence of 14 bp at the most prominent hypersensitive site (-405) is closely related to sequences 5’ to several other eucaryotic genes. Introduction Analyses of eucaryotic gene expression must take into account the changes in chromatin organization that occur when a gene becomes active. Two aspects of these chromatin structural changes can be detected following digestion with DNAase I. First, there is a preferential sensitivity to digestion that extends throughout the transcription unit. This overall sensitivity is specific to cells in which the gene is expressed (Garel and Axel, 1976; Weintraub and Groudine, 1976) and, in the case of chicken globin genes, results from the association of two nuclear proteins, HMG 14 and HMG 17, with nucleosomes of the region (Weisbrod et al., 1980). Second, there are very local alterations in chromatin structure which result in discrete sites that are extremely sensitive to DNAase I cleavage (Wu et al., 1979; Stalder et al., 1980; Wu, 1980). Many, but not all, of these DNAase l-hypersensitive
DNAase l-Hypersensitive Glue Protein Gene, Sgs4 sites are clustered at the 5’ side of genes (Kuo et al., 1979; Stalder et al., 1980; Wu, 1980; Herbomel et al., 1981). For the /I globin (Stalder et al., 1980) and preproinsulin (Wu and Gilbert, 1981) genes, these hypersensitive sites were shown to be tissue-specific, and the P-globin sites are missing from the embryonic precursor cells. Thus their presence coincides with gene expression. We have chosen to examine the DNAase-hypersensitive sites near the Drosophila glue protein gene, Sgs4. The glue protein genes are a group of at least eight genes that are expressed during the third instar of larval development in the salivary glands of Drosophila larvae (Korge, 1975; Beckendorf and Kafatos, 1976). At the end of the third instar the proteins coded by these genes are used as a glue to attach the puparium to a dry surface (Fraenkel and Brookes, 1953). The Sgs4 gene was chosen for this study because its DNA sequence organization had been studied (McGinnis et al., 1980; Muskavitch and Hogness, 1980) and because numerous mutants had been identified that produce little or no SGS-4 protein (Korge, 1975, 1977; Beckendorf and Kafatos, 1976) or RNA (Muskavitch and Hogness, 1980). We report that a complex of five tissue-specific hypersensitive sites is present 5’ to Sgs4 in chromatin from third instar salivary glands. Analysis of two mutant strains shows that formation of this complex depends on one or both of the two distal sites or on the DNA sequences near them. Similarly, at least two of the sites or their adjoining DNA sequences are required for normal expression of Sgs4. We also point out that there is a sequence associated with the most prominent hypersensitive site that is found near several other eucaryotic genes. Results DNAase-Hypersensitive Sites near Sgs4 in Embryonic Nuclei To search for DNAase-hypersensitive sites near a transcriptionally inactive Sgs4 gene, we briefly treated nuclei from 6-l 8 hr embryos with DNAase I, extracted the DNA and cut it with the restriction endonuclease Sst I, which produces a 15 kb fragment containing Sgs4 and flanking regions on both sides (Figure 1). The digested DNA was then separated on a 0.9% agarose gel, transferred to DBM paper (Alwine et al., 1977) and hybridized to a 32P-labeled 1.1 kb fragment complementary to the left end of the large Sst fragment. As first pointed out by Wu (1980), this procedure indirectly labels one end of the 15 kb fragment and any fragments produced by DNAase digestion in this region. DNAase-hypersensitive sites will then appear on the autoradiograph as discrete bands whose size corresponds to the distance of the hypersensitive site from the labeled end. As shown in Figure 2a, one minor and two major DNAase-hypersensitive sites can
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be detected within the 15 kb Sst fragment (from embryonic nuclei). These sites are located 2.1, 3.5 and 4.6 kb 3’ to the transcribed region of Sgs4. No hypersensitive sites are detected adjacent to the gene or in the 4 kb 5’ to the gene. Similar experiments with nuclei from the Schneider 2 cell line (Schneider, 1972) have revealed the same three 3’ sites and no others (data not shown). The sites identified in this way result from the chromatin conformation and not from an inherent preference of DNAase for a particular base sequence. When the substrate for DNAase digestion is naked DNA rather than nuclei, no prominent bands can be detected within Sgs4 or on either side of it (Figure 2b). DNAase Hypersensitive Sites near Sgs4 in Salivary Glands To determine whether the pattern of sites changes when Sgs4 is expressed, we mass-isolated salivary glands from mid-third instar larvae. To be sure that the glands came from larvae of the appropriate stage, some glands were squashed, and their polytene chromosomes were examined for puffed loci. The intermolt puffs 3Cl l-l 2 and 68C, which make major components of the glue (Korge, 1977; Akam et al., 19781, were visible, whereas the 74EF and 75B puffs, early members of the larval ecdysone-induced series (Ashburner, 19721, were not. Petri et al. (1977) have reported that during mass isolation of Drosophila tissues the heat-shock response may be induced. However, the heat-shock puffs at 87A and 87C were not visible in the chromosomes of our mass-isolated glands. Thus we conclude that the mass-isolated salivary glands are suitable for studies of Sgs4 chromatin structure. Nuclei from these glands were then digested briefly with DNAase I and analyzed as described above. The restriction enzyme used in the secondary digestion was Sal I, which produces a 19 kb restriction fragment whose left end is 2.45 kb 5’ to Sgs4 (Figure 1). When this is probed from the left end, a complex series of XdDml554 --
DNAase l-hypersensitive sites can be detected (Figure 3). The most prominent site is located about 400 bp 5’ to the Sgs4 cap site. Adjacent to this and extending approximately to -300 is a region that in this sample appears uniformly sensitive to DNAase I. The profile of this region is variable, appearing on some samples as a more defined peak at -330 (data not shown). Somewhat less sensitive DNAase I sites appear at -480, -70 and +30 (Figures 3 and 4). Near the top of the gel, the same 3’-hypersensitive sites seen in embryos can be observed. Because this experiment measured from the Sal I site 2-2.5 kb away, it located the hypersensitive sites with a resolution of +30-50 bp. We have been able to locate the most prominent site more accurately by following the DNAase treatment with digestion by Barn HI and Eco RI. As shown in Figure 1, this produces a fragment of 800 bp whose right end coincides with the right end of the gene (M. Muskavitch and D. Hogness, submitted). We then determined the position of the most prominent hypersensitive site in this interval by using as a probe either the Xho I-Eco RI fragment from the right end or the Barn HI-Xho I fragment from the left end. Both the Eco RI-Xho I fragment and the Eco RI-Sau 3A fragment were used as size standards, since they flank the DNAase site and their lengths have been determined by DNA sequencing (M. Muskavitch and D. Hogness, submitted). A densitometer tracing of one lane from this gel is
+10 F
I-
0
pDmRl5
,'
--_
,'
2
a
-5 I
1 kb
-.._
Figure 2. DNAase-Hypersensitive Chromatin and in Purified DNA 200t’p
Figure
1. Partial
Restriction
Map of the Sgs4 Region
(4) Transcribed region of Sgs4. Brackets above the map show the extent of genomic DNA cloned in hdDml554and pDmRl.5. For clarity only those restriction sites used in the present analysis are shown. More complete maps can be found in Muskavitch and Hogness (1980: submitted) and in McGinnis et al. (1980).
Sites
near
Sgs4
in Embryonic
(a) Embryonic nuclei were isolated and treated for 3 min with 0 or 15.6 units of DNAase I per milliliter. The DNA was then extracted, digested to completion with Sst I and analyzed by Southern blotting (Southern, 1975). with the use of the 1 .l kb Barn fragment isolated from AdDml554 d) as a probe. (b) Purified DNA rather than nuclei was treated for 75 set with 0.1 or 0.2 units of DNAase I per milliliter, digested with Sst I and analyzed as in (a). Thin lines connect corresponding regions of the diagram and the two gels. (h) DNAasehypersensitive sites.
Interacting 603
DNAase
l-Hypersensitive
ORE -
Sites
ORE+
HAS +
BER+ _ il
Figure 4. Summary of the Hypersensitive Sites Upstream from Sgs4 and Their Relation to the Deletions in the Oriental and BER 1 Strains Only the 5’ end of Sgs4 is shown. (9) Hypersensitive mentioned in the text, the site at -330 sometimes appears region (bracket). Solid bars below line: the two deletions.
sites. As as a broad
n
Figure 5. The Major DNAase bp Upstream from Sgs4
Figure 3. DNAase-Hypersensitive Mutant Salivary Gland Chromatin
Sites near Sgs4
in Wild-type
and
Salivary gland nuclei were isolated and treated for 3 min with DNAase I (0. 7.8, 7.8 or 5.2 U/ml for the four samples shown). After DNA extraction, the samples were digested with Sal I and analyzed by Southern blotting. The probe was the 0.31 kb Barn HI fragment isolated from pDmRl.5 (Figure 1). (+ and -) Treated or untreated with DNAase I. (p Position corresponding to the transcribed region of Sgs4. Locations of DNAase l-generated fragments 5’ to Sgs4 in the ORE+ and HAS+ lanes are indicated between the gel tracks. The bands near the top of the gel correspond to the 3’ hypersensitive sites. Their locations vary among the three strains as a result of differences in the length of the coding sequence (M. Muskavitch and D. Hogness. submitted) and of the 3’ flanking sequences.
shown in Figure 5. It locates the prominent -405 +5 bp from the Eco RI site.
site at
DNAase Hypersensitive Sites in Sgs4 Mutants We reasoned that the possible role of the Y-hypersensitive sites in Sgs4 expression might be clarified by analysis of SgsGdefective mutants, two groups of which have recently been characterized by Muskavitch and Hogness (1980; submitted). Strains in the Oriental group accumulate l-3% as much Sgs4 RNA as the Sgs4+ standard strain, Oregon R, and are missing 52 bp between -307 and -358 (M. Muskavitch and D. Hogness, submitted). The BER 1 strain does not produce detectable amounts of Sgs4 RNA and has a deletion of 103 bp between -394 and -496 (M. Muskavitch and D. Hogness, submitted). We have determined the pattern of DNAase-hypersen-
l-Hypersensitive
Site Is Located
405
DNA from salivary gland nuclei that had been treated with 7.8 units DNAase I/ml was digested with Barn HI and Eco RI and analyzed by Southern blotting with use of the 391 bp Xho I-Eco RI fragment that lies just 5’ to Sgs4 as the probe (see diagram above and Figure 1). The resulting autoradiograph was traced on a Joyce Loebl microdensitometer, and the position of the hypersensitive site was compared with standards from pBR322 and from pDmR1 S. a pBR322 derivative that contains the 1.5 kb Eco RI fragment that adjoins Sgs4 on the 5’ side. The two Drosophila fragments that flank the hypersensitive site are Xho I-Eco RI (391 bp) and Sau SA-(Eco RI (443 bp).
sitive sites near Sgs4 in salivary gland nuclei from an Oriental strain called Hikone AS and in BER 1. This pattern is distinctly altered in both cases. In Hikone AS, most of the 5’ sites are the same as those of Oregon R, except that the region of hypersensitivity extending from -400 to -300 is truncated at about -350, corresponding to the location of the deletion (Figure 3). Because the deletion is located between the labeled end of the restriction fragment and the two hypersensitive sites near the end of the gene (-70 and +30), these two appear to be displaced by about 50 bp in the 5’ direction. Their position relative to the gene, however, remains the same. Therefore, while the deletion removes a region that normally becomes hypersensitive, it does not affect the pattern of sensitivity in adjacent regions. There may, however, be an alteration in the relative frequencies with which the remaining sites are cut. The -70 and +30 sites appear to be underrepresented in the Hikone AS chromatin. The pattern is more drastically altered in BER 1.
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Despite the fact that only the sequences corresponding to the two most distal sites are missing, no hypersensitive sites can be detected in BER 1 salivary gland nuclei in the region 5’ to Sgs4 (Figure 3). Thus the missing sequences appear to be required for downstream alterations in chromatin structure, as well as for expression of the gene. Discussion These results show that when Sgs4 becomes active in larval salivary glands, the chromatin structure 5’ to the gene is extensively altered so that five regions become accessible to DNAase I. In contrast, by the criterion of DNAase I hypersensitivity, the region 3’ to the gene is unaltered. A complex of hypersensitive sites has also been described in the region 5’ to several other active genes, including the Drosophila heat-shock genes (Wu, 1980; Keene et al., 1981), the rat preproinsulin gene (Wu and Gilbert, 1981) and the genes of polyoma virus (Herbomel et al., 1981). The availability of Sgs4 mutants has enabled us to learn something about the involvement of some parts of the complex in Sgs4 expression. In Hikone AS, the region of hypersensitivity corresponding to the DNA deletion is simply missing; the remainder of the hypersensitive sites are present and located over the same regions of DNA as in Oregon R. This result suggests that the deleted hypersensitive region is required for normal Sgs4 expression-without it, accumulation of Sgs4 message is reduced 50fold (M. Muskavitch and D. Hogness, submitted). This result also rules out a possible explanation for the formation of the hypersensitive sites. It might have been that sites appear at a defined distance from an active gene, regardless of the sequence located there. On such a model, the location of the sites might be dictated by steric or topological constraints on chromosome folding or supercoiling as the gene became accessible for transcription. Our results for Sgs4 are inconsistent with such a model, since the sites at -405 and -480 form over their normal sequences in Hikone AS, even though those sequences are 52 bp closer to the gene than they are in Oregon Ft. The second type of deletion mutant, BER 1, makes no detectable Sgs4 RNA and is missing a region of DNA that normally contains two hypersensitive sites, -405 and -480. In BER 1, not only are these sites missing, but also the other three 5’ sites are missing. This result implies that there are interactions among the various chromatin regions 5’ to Sgs4 and that they are hierarchical. Appearance of the three Sgs4 proximal sites is dependent on the sequences missing in BER 1, perhaps on the change in conformation which we detect as DNAase hypersensitivity. Although additional mutants will be required to distinguish between the -405 and -480 sites, we favor the idea
that the very prominent site at -405 is the one on which the remainder of the region is dependent. What kind of sequences correspond to the hypersensitive sites? There are many clusters of A-T-rich sequence 5’ to Sgs4 (M. Muskavitch and D. Hogness, submitted), and to the degree that we have localized the hypersensitive sites, all might be located in such clusters. However, we can rule out base composition as the sole criterion for hypersensitivity. There are many additional A-T-rich regions 5’ to Sgs4, most notably those between -100 and -300, which are flanked by hypersensitive sites but are not themselves sensitive. Several of the hypersensitive sites are located at or near specific sequences that may be important for gene expression. Two of the hypersensitive sites (- 70 and +30) are located near the beginning of the transcribed region. From comparison of many eucaryotic genes, three kinds of conserved sequences have been detected in this region. The sequence at the point of transcription initiation and attachment of the cap structure usually consists of an A residue surrounded by pyrimidines (Breathnach and Chambon, 1981). The Goldberg-Hogness or TATAA box approximately 23-30 bp 5’ to the cap site is very highly conserved and has been implicated in accurate transcription initiation (Goldberg, 1979; Grosschedl and Birnsteil, 1980; Benoist and Chambon, 1981; Grosveld et al., 1981; Mathis and Chambon, 1981). Benoist et al. (1980) have identified a third conserved sequence, GGPyCAATCT, often located 68-80 bp 5’ to eucaryotic genes. The region around this site has been shown to be essential for transcription of the herpes virus thymidine kinase gene (McKnight et al., 1981). Sgs4 has examples of all three of these sequences (M. Muskavitch and D. Hogness, submitted). Because of uncertainty regarding its exact location, the hypersensitive site that we have placed at -70 might correspond to either the GGPyCAATCT sequence or to TATAA. Similarly, the +30 hypersensitive site might correspond to the cap site. The three remaining hypersensitive sites are located 300-500 bp from the gene, in a region that has not been studied for many genes. However, there is some precedent for regulatory effects of sequences relatively far from the genes they control. Transcription in vivo of SV40 early genes requires sequences within the 72 bp direct repeats that are located 122-266 bp upstream (Benoist and Chambon, 1981; Gruss et al., 1981). In addition, transcription of the sea urchin H2A gene can be modulated 60 to 100 fold by loss or inversion of a segment normally located between -184 and -524 (Grosschedl and Birnsteil, 1980). The three Sgs4-hypersensitive sites are located over potentially interesting sequences. The -480 site corresponds to a threefold direct repeat and the -330 site to a partially selfcomplementary inverted repeat
interacting 605
DNAase
Table 1. Sequences
I-Hypersensitive
Located
Sites
5’ to Several
Drosophila
Genes Are Similar to the Sgs4 Sequence
Gene
Sequences
Distance Gene
sgs4
AAACTAAAGCTGGT
396 323/l
68C group
ll/lll
AAA.
68C group
IV
TAAAGCGCCT
References M. Muskavitch 57
M. Garfinkel. ,‘
AATATAAAGCATAT
420
hsp70
87A
AAA.
235
lngolia
hsp70
87C
AAAATAAAGCGAAT
235
Torok
copia
TAAAGAATAT
AATATAAAG.
TAGT
at -405
to and D. Hogness.
submitted
R. Pruitt and E. Meyerowitz,
et al. (1980); and Karch
Torok
and Karch
personal
communication
(1980)
(1980)
Rubin et al. (1980)
Five prominent RNAs have been identified in larval D. melanogaster salivary glands during the intermolt period (Wolfner. 1980). One of these is encoded by Sgs4 (McGinnis et al., 1980; Muskavitch and Hogness, 1980). Three others, called groups II. Ill and IV. are encoded by a cluster of genes located at 68C on the polytene chromosome map (M. Meyerowitz and D. Hogness. submitted). Two of these genes, II and Ill. are divergently transcribed, and the second sequence shown above is located between them. The third sequence is located 5’ to the group IV gene. Copies of the major D. melanogaster heat-shock gene, hsp70. are located at both 87A and 87C (Ashburner and Banner. 1979). Because the genes are slightly divergent at the two loci (Torok and Karch. 1980). one from each has been included. The copia sequence is located within the terminal direct repeat between positions 45 and 58 (Rubin et al., 1980).
(M. Muskavitch and D. Hogness, submitted). Perhaps the most interesting site is that centered at -405. It is the most sensitive to DNAase cleavage, and as discussed above, may be required for formation of the other sites and for transcription of the gene. Between -396 and -409 there is a 14 bp sequence that shares homology with sequences 5’ to the glue protein genes at 68C on the third chromosome and with sequences 5’ to copies of the heat-shock gene hsp70 located at bands 87A and 87C (Table 1). There is also a very similar sequence in the terminal direct repeat of the transposable element, copia. Whether in these other cases this sequence is involved in DNAase I hypersensitivity or is required for gene expression is not yet known. However, it seems unlikely to have occurred 5’ to all these genes by chance. In a random sequence, the invariant part of the sequence would be expected about once every 16 kb. In addition, a related sequence, GNNAAGNANNNAT, is found in the 72-nucleotide repeats of SV40 and the long terminal repeats of several mammalian retroviruses (S. K. Beckendorf, unpublished). Both the SV40 repeats and the retroviral long terminal repeats show DNAase l-hypersensitive sites when the associated genes are active (C. Cremisi and H. Weintraub, personal communication; Groudine et al., 1981). The SV40-hypersensitive site has been located within 15 bp of the conserved sequence. In conclusion, examination of the chromatin surrounding Sgs4 has revealed eight hypersensitive sites, five of which are tissue-specific. At least three of the five depend for their appearance on a region containing the most prominent site, which is located about 405 bp 5’ to the gene. This region also appears to be necessary for Sgs4 expression. Sequences related to that of the prominent hypersensitive site are found upstream from several other genes, suggesting that they are similarly dependent on DNAase Ihypersensitive regions.
Experimental
Procedures
Strains of Drosophila The Oregon R wild-type strain of D. melanogaster (obtained from W. Petri, Boston College) was used as the standard for DNA sequence arrangement near Sgs4. The two Sgs4 mutant strains used are Hikone AS (HAS; Bowling Green University) and Berkeley 1 (BER 1, originally identified as a laboratory stock that made no Sgs4 protein). The X chromosomes of these stocks were made isogenic before the experiments reported here were conducted. Collection of Embryos Embryos 6-18 hr old were obtained from a laboratory population of D. melanogaster (Oregon R) maintained at 25’C. as described by Elgin and Miller(l978). Embryos were washed extensively with water, dechorionated by stirring in 50% Clorox for 1 min and washed with distilled water, 70% ethanol and more distilled water. They were then quick-frozen at -8O’C until used. Salivary Gland Isolation Salivary glands were isolated from mid-third instar by a modification of the methods of Zweidler and Cohen (1971) and Boyd (1978). Briefly, larvae are reared en masse in disposable plastic dishes (Elgin and Miller, 1978) and isolated in late third instar from the walls of the dishes. The larvae are thoroughly washed to remove yeast and food particles and then suspended in organ medium: 25 mM disodium glycerophosphate; 10 mM KH,POI; 30 mM KCI; 10 mM MgC12. 3 mM CaCb and 162 mM sucrose (Cohen and Gotchel. 1971). All subsequent steps are carried out at 4’C in organ medium. The larvae are then disrupted by passage between two rotating stainless steel rollers that are spaced 0.18 mm apart. Large debris such as empty carcasses is removed by passage through Nitex screen (1 mm* pore size). Salivary glands are then purified by repeated settlings at unit gravity, centrifugal agitation (Zweidler and Cohen, 1971) and sedimentation through a 4 X 80 cm column containing a 5-10% gradient of Ficoll in organ medium. Most contaminating tissue remaining after these steps (mainly gut and Malpighian tubules) is removed through a drawn out Pasteur pipet attached to an aspirator. This procedure results in 90-95% pure salivary glands with a yield of 0.5-l ml per 200 g larvae. Isolation of Embryonic Nuclei Dechorionated embryos (5 g) were ground with a mortar and pestle at -70°C. The powdered embryos were then stirred into 20 ml of buffer A plus 0.25 M sucrose at 0°C (buffer A is 60 mM KCI, 15 mM NaCI. 1 mM EDTA. 0.1 mM EGTA, 0.15 mM spermine, 0.5 mM spermidine, 15 mM Tris-HCI [pH 7.41, 0.5 mM dithiothreitol and 0.1 mM phenylmethylsulfonyl fluoride [PMSF]: Wu et al., 1979) and
Cell 606
homogenized in a Dounce homogenizer fitted with a B pestle (six to eight strokes). The homogenate was filtered through 100 pm pore size Nitex screen, and the filtrate was centrifuged for 1 min at 2000 rpm (650 x g) in a Sorvall HB4 rotor. After discarding the pellet, the supernatant was brought to 0.2% Nonidet-P40. vortexed vigorously and centrifuged for 10 min at 6000 rpm (5860 X g) in the same rotor to pellet crude nuclei. The pellet was resuspended in buffer A plus 0.25 M sucrose and layered onto a crude gradient prepared from buffer A* plus 0.85 M sucrose and buffer A’ plus 1.7 M sucrose. (Buffer A* is buffer A without EDTA and EGTA.) The gradient was centrifuged for 20 min at 10,500 rpm (18,000 X g). The supernatant was removed, and the purified nuclear pellet was resuspended in nuclear buffer (60 mM KCI. 15 mM NaCI, 0.05 mM Car&, 0.1 mM EDTA, 15 mM Tris-HCI [pH 7.41. 0.5 mM dithiothreitol and 0.25 M sucrose). This procedure yielded nuclei containing about 0.5 mg DNA from each gram of embryos. Isolation of Salivary Gland Nuclei Two milliliters of purified salivary glands were suspended in 10 ml buffer A plus 0.25 M sucrose in a Dounce homogenizer and homogenized with four to eight strokes of an A pestle. Nonidet-P40 (10%; 250 Al) was added, and the suspension was vortexed until almost all cells had lysed, but nuclei were still intact. The suspension was strained through 110 Am Nitex and then centrifuged for 2 min at 1000 rpm in the HB4 rotor (164 X g). The crude nuclear pellet was then purified on the same sucrose gradient described for the embryonic nuclei. The resulting purified nuclei were resuspended in nuclear buffer. One milliliter of settled salivary glands yielded nuclei containing 100-l 50 Ag DNA. DNAase I Digestion DNAase I (DPFF grade) was purchased from Worthington. Just prior to use it was diluted in 60 mM KCI. 15 mM NaCI. 100 mM MgCI?, 0.1 mM CaC12. 15 mM Tris-HCI (pH 7.4) and 0.5 mM dithiothreitol. Digestions of intact nuclei were initiated by adding 25 Al of the appropriate DNAase I dilution to 475 pl of nuclear suspension at 25°C. After 3 min the digestion was stopped by adding 20 pl of 0.5 M EDTA. DNA Extraction Nuclei were lysed by addition of SDS to 0.5% and treated overnight at 37°C with proteinase K (E. Merck, Beckman) at 70 pg/ml. Each sample was extracted once with chloroform-isoamyl alcohol (24:1, v/v); once with equal volumes of chloroform-isoamyl alcohol and phenol: and once with ether. One volume of water was added, and RNA was removed by treatment with RNAase A and RNAase Tl (heattreated at 8O’C for 30 min to inactivate DNAases; final concentrations were 50 pgs/ml for RNAase A and 37 U/ml for RNAase Tl) at 30°C for 2 hr for 15 min. RNAases were removed by proteinase K treatment for 3 hr in 0.5% SDS at 37°C. and the protease was removed by a repetition of the organic extractions. The final aqueous phase was made 0.2 M in sodium acetate, and DNA was precipitated overnight at -20°C in 2.5 volumes ethanol. Preparation of Nick-Translated Probes Specific restriction fragments containing DNA from the Sgs4 region were isolated from the A derivative hdDm1554 (Muskavitch and Hogness. 1980) or the pBR322 derivative pDmRl.5, which was constructed by inserting the 1.5 kb Eco RI fragment located immediately 5’ to Sgs4 into the Eco RI site of pBR322. The extent of genomic DNA included in these clones is indicated in Figure 1. Afler appropriate restriction, the cloned DNAs were subjected to electrophoresis on agarose gels and visualized by ethidium-bromide staining. The desired fragment was excised from the gel and recovered by the glass fiber filter method of Davis et al. (1980). The fragments were then nick-translated according to the procedure of Maniatis et al. (1975). Southern Hybridizations Restriction endonucleases were obtained from New England BioLabs or Bethesda Research Laboratories and used according to the sup-
plier’s directions. Five micrograms of genomic DNA was digested per slot and fractionated on horizontal 0.556-l .O% agarose gels. Electrophoresis buffer was the Tris-borate-EDTA buffer described by Peacock and Dingman (1968). DNA fragments were transferred to diazotized paper, hybridized to nick-translated probe and washed by the method of Wahl et al. (1979). Autoradiography was performed at -8O’C by using a Du Pont Cronex Lightning Plus intensifying screen and Kodak XR-5 film. Before a previously probed DNA paper was reused, the old labeled probe was washed off by gently shaking the paper in 0.4 M NaOH for 15 min at room temperature and then rinsing thoroughly with distilled water. Acknowledgments We would like to thank Marc Muskavitch and David Hogness for continual communication of their results on Sgs4 organization and for providing us with several Sgs4 cloned sequences. We are grateful to Carl Wu and Sarah Elgin for instruction in DNAase digestion of chromatin; to Lawrence McGahey for preparation of nitrobenzyloxymethyl pyridinum chloride: and to Andrew Cockburn, John Farrell, Jr., Martha Fedor and William McGinnis for critically reading the manuscript. This research was supported in part by a Basil O’Connor Starter Research Grant from the March of Dimes Birth Defects Foundation and by a grant from the National Institute of Child Health and Human Development. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received
November
13. 1981;
Released
March
5, 1981
References Alwine, J. C., Kemp, D. J. and Stark, G. R. (1977). Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc. Nat. Acad. Sci. USA 74, 5350-5354. Akam. M.. Roberts, D.. Richards, Drosophila: the genetics of two 215-225.
G. and Ashburner. M. (1978). major larval proteins. Cell 13,
Ashburner, M. (1972). Puffing patterns in Drosophila melanogaster and related species. In Results and Problems in Cell Differentiation 4. W. Beermann, ed. (New York and Berlin: Springer-Verlag), pp. 101-151. Ashburner. M. and Bonner. J. J. (1979). The induction in Drosophila by heat shock. Cell 7 7, 241-254.
of gene activity
Beckendorf. S. K. and Kafatos, F. C. (1976). Differentiation in the salivary glands of Drosophila melanogaster: characterization of the glue proteins and their developmental appearance. Cell 9, 365-373. Benoist, C. and region: sequence
Chambon, P. (1981). The SV40 early promoter requirements in viva. Nature 290. 304-310.
Benoist, C.. O’Hare, K., Breathnach, R. and Chambon. P. (1980). The ovalbumin gene-sequence of putative control regions. Nucl. Acids Res. 8, 127-l 42. Boyd. J. B. (1978). Mass isolation of salivary glands from larvae of D. hydei. In The Genetics and Biology of Drosophila, M. Ashburner and T. R. F. Wright, eds. (New York: Academic Press) 2a, pp. 127-l 35. Breathnach, R. and Chambon. P. (1981). Organization and expression of eukaryotic split genes coding for proteins. Ann. Rev. Biochem. 50, 349-383. Cohe?. L. H. and Gotchel. B. V. (1971). Histones of polytene nonpolytene nuclei of Drosophila melanogaster. J. Biol. Chem. 1841-l 840. Davis, R. W., Botstein. D. and Roth, J. R. (1980). Genetic Engineering: Advanced Bacterial Genetics. Harbor, New York: Cold Spring Harbor Laboratory).
and 246,
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Elgin. S. C. R. and Miller, D. W. (1978). Mass rearing of flies and mass production and harvesting of embryos. In The Genetics and Biology of Drosophila, M. Ashburner and T. R. F. Wright, eds. (New York: Academic Press) 2a, pp. 112-l 21.
Rubin. G. M., Brorein. W. J.. Jr., Dunsmuir, P., Flavell, A. J., Levis, R. Strobel. E.. Toole, J. J. and Young, E. (1980). “Copia-like” transposable elements in the Drosophila genome. Cold Spring Harbor Symp. Ouant. Biol. 45, 619-628.
Fraenkel. G. and Brookes, V. J. (1953). The process by which the puparia of many species of flies become fixed to a substrate. Biol. Bull. 105, 442-449.
Schneider, Drosophila
Garel. A. and Axel, R. (1976). Selective digestion of transcriptionally active ovalbumin genes from oviduct nuclei. Proc. Nat. Acad. Sci. USA 73, 3966-3970. Goldberg, California.
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E. M. (1975). Detection of specific sequences among DNA separated by gel electrophoresis. J. Mol. Biol. 98,
Stalder. J.. Larsen, A., Engel, J. D.. Dolan, M., Groudine, M. and Weintraub. H. (1980). Tissue-specific DNA cleavages in the globin chromatin domain introduced by DNAase I. Cell 20, 451-460.
Grosschedl, R. and Birnsteil. ht. L. (1980). Spacer DNA sequences upstream of the TATAAATA sequence are essential for the promotion of H2A histone gene transcription in viva. Proc. Nat. Acad. Sci. USA 77, 7102-7106.
Torok, I. and Karch. F. (1980). Nucleotide sequences of heat shock activated genes in Drosophila melanogaster. I. Sequences in the region of the 5’ and 3’ ends of the hsp 70 gene in the hybrid plasmid 56H8. Nucl. Acids Res. 8, 3105-3123.
Grosveld, G. C., Shewmaker. C. K., Jat, P. and Flavell. R. A. (1981). Localization of DNA sequences necessary for transcription of the rabbit P-globin gene in vitro. Cell 25, 215-226.
Wahl. G. M.. Stern, M. and Stark, G. R. (1979). Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl paper and rapid hybridization by using dextran sulfate. Proc. Nat. Acad. Sci. USA 76, 3683-3687.
Gruss, P.. Dhar. R. and Khoury. G. (1981). Simian virus 40 tandem repeated sequences as an element of the early promoter. Proc. Nat. Acad. Sci. 78, 943-947. Herbomel, P., Saragosti. S.. Blangy, D. and Yaniv, M. (1981). Fine structure of the origin-proximal DNAase l-hypersensitive region in wild-type and EC mutant polyoma. Cell 25, 651-658. Ingolia, T. D.. Craig, E. A. and McCarthy, B. J. (1980). Sequence of three copies of the gene for the major Drosophila heat shock induced protein and their flanking regions. Cell 27, 669-679. Keene. M. A., Corces, V.. Lowenhaupt, K. and Elgin, S. C. R. (1981). DNAase I hypersensitive sites in Drosophila chromatin occur at the 5’ ends of regions of transcription. Proc. Nat. Acad. Sci. USA 78, 143-146.
Wolfner, M. (1980). Ecdysone-responsive genes of the salivary glands of Drosophila melanogaster. Ph.D. thesis, Stanford University, Stanford, California. Weintraub. H. and Groudine, H. (1976). active genes have an altered conformation.
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Korge. G. (1975). Chromosome puff activity and protein synthesis in larval salivary glands of Drosophila melanogaster. Proc. Nat, Acad. Sci. USA 72, 4550-4554.
Wu, C.. Bingham, P. M.. Livak. K. J., Holmgren. R. and Elgin. S. C. R. (1979). The chromatin structure of specific genes: I. Evidence for higher order domains of defined DNA sequence. Cell 76, 797-806.
Korge, G. (1977). Direct correlation between a chromosome puff and the synthesis of a larval saliva protein in Drosophila melanogaster, Chromosoma 62, 155-l 74.
Zweidler. A. and Cohen, L. H. (1971). Large scale isolation and fractionation of organs of Drosophila melanogaster larvae. J. Cell Biol. 51, 240-248.
Kuo. M. T.. Mandel, J. L. and Chambon, P. (1979). DNA methylation correlation with DNAase I sensitivity of chicken ovalbumin and conalbumin chromatin. Nucl. Acids Res. 7, 2105-2113. Maniatis. T.. Jeffrey, A. and Kleid, D. G. (1975). Nucleotide sequence of the rightward operator of phage A. Proc. Nat. Acad. Sci. USA 72, 1184-1188. Mathis. D. J. and Chambon, box is required for accurate 290, 31 o-31 5.
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McGinnis, W.. Farrell. J., Jr., and Beckendorf. S. K. (1980). Molecular limits on the size of a genetic locus in Drosophila melanogaster. Proc. Nat. Acad. Sci. USA 77, 7367-7371. McKnight. S. L.. Gavis, E. R., Kingsbury, Ft. and Axel. R. (1981). Analysis of transcriptional regulatory signals of the HSV thymidine kinase gene: identification of an upstream control region. Cell 25, 385-398. Meyerowitz, a Drosophila
E. and Hogness. D. S. (1982). Molecular organization puff site that responds to ecdysone. Cell 28, 165-t
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Peacock. A. C. and Dingman, C. W. (1968). Molecular weight estimation and separation of ribonucleic acid by electrophoresis in agarose-acrylamide composite gels. Biochemistry 7, 668-674. Petri, W. H.. Wyman, A. R. and Henikoff. S. (1977). Synthesis of “heat shock” mRNA by Drosophila melanogaster follicle cells under standard organ culture conditions. Drosophila Information Service 52, 80.
Note Added
in Proof
The article referred to as “M. Muskavitch and D. Hogness, is now in press for the July 1980 issue of Cell.
submitted”
1962
by MIT
A Complex of Interacting Sites near the Drosophila Antony W. Shermoen and Steven Department of Molecular Biology University of California Berkeley, California 94720
K. Beckendorf
Summary The chromatin structure adjacent to the Drosophila glue protein gene Sgs4 changes drastically when the gene is active. In nuclei from embryos or tissue culture cells in which Sgs4 is inactive, there are three DNAase l-hypersensitive sites 3’ to the gene, but none near its 5’ end. In the nuclei of late third instar salivary glands, Sgs4 is actively transcribed, and a complex of five DNAase l-hypersensitive sites appears 5’ to the gene. Two of the sites are near the point of transcription initiation, at -70 and +30. The others are much farther from the gene at -330, -405 and -480 and are affected by small deletions: one deletion reduces expression about 50fold and removes sequences corresponding to the -330 hypersensitive site; another makes no Sgs4 RNA and removes sequences corresponding to two hypersensitive sites, -405 and -480. Thus the hypersensitive sites, or DNA sequences within 50 bp of them, seem to be required for normal gene expression. Distinct changes are seen in the chromatin from salivary glands of these mutant strains. The first strain is simply missing the -330 hypersensitive site, while the second is missing all of the tissue-specific 5’ sites, even though sequences corresponding to three of them remain. This suggests that hierarchical interactions among the regions 5’ to Sgs4 are required for its full expression. A sequence of 14 bp at the most prominent hypersensitive site (-405) is closely related to sequences 5’ to several other eucaryotic genes. Introduction Analyses of eucaryotic gene expression must take into account the changes in chromatin organization that occur when a gene becomes active. Two aspects of these chromatin structural changes can be detected following digestion with DNAase I. First, there is a preferential sensitivity to digestion that extends throughout the transcription unit. This overall sensitivity is specific to cells in which the gene is expressed (Garel and Axel, 1976; Weintraub and Groudine, 1976) and, in the case of chicken globin genes, results from the association of two nuclear proteins, HMG 14 and HMG 17, with nucleosomes of the region (Weisbrod et al., 1980). Second, there are very local alterations in chromatin structure which result in discrete sites that are extremely sensitive to DNAase I cleavage (Wu et al., 1979; Stalder et al., 1980; Wu, 1980). Many, but not all, of these DNAase l-hypersensitive
DNAase l-Hypersensitive Glue Protein Gene, Sgs4 sites are clustered at the 5’ side of genes (Kuo et al., 1979; Stalder et al., 1980; Wu, 1980; Herbomel et al., 1981). For the /I globin (Stalder et al., 1980) and preproinsulin (Wu and Gilbert, 1981) genes, these hypersensitive sites were shown to be tissue-specific, and the P-globin sites are missing from the embryonic precursor cells. Thus their presence coincides with gene expression. We have chosen to examine the DNAase-hypersensitive sites near the Drosophila glue protein gene, Sgs4. The glue protein genes are a group of at least eight genes that are expressed during the third instar of larval development in the salivary glands of Drosophila larvae (Korge, 1975; Beckendorf and Kafatos, 1976). At the end of the third instar the proteins coded by these genes are used as a glue to attach the puparium to a dry surface (Fraenkel and Brookes, 1953). The Sgs4 gene was chosen for this study because its DNA sequence organization had been studied (McGinnis et al., 1980; Muskavitch and Hogness, 1980) and because numerous mutants had been identified that produce little or no SGS-4 protein (Korge, 1975, 1977; Beckendorf and Kafatos, 1976) or RNA (Muskavitch and Hogness, 1980). We report that a complex of five tissue-specific hypersensitive sites is present 5’ to Sgs4 in chromatin from third instar salivary glands. Analysis of two mutant strains shows that formation of this complex depends on one or both of the two distal sites or on the DNA sequences near them. Similarly, at least two of the sites or their adjoining DNA sequences are required for normal expression of Sgs4. We also point out that there is a sequence associated with the most prominent hypersensitive site that is found near several other eucaryotic genes. Results DNAase-Hypersensitive Sites near Sgs4 in Embryonic Nuclei To search for DNAase-hypersensitive sites near a transcriptionally inactive Sgs4 gene, we briefly treated nuclei from 6-l 8 hr embryos with DNAase I, extracted the DNA and cut it with the restriction endonuclease Sst I, which produces a 15 kb fragment containing Sgs4 and flanking regions on both sides (Figure 1). The digested DNA was then separated on a 0.9% agarose gel, transferred to DBM paper (Alwine et al., 1977) and hybridized to a 32P-labeled 1.1 kb fragment complementary to the left end of the large Sst fragment. As first pointed out by Wu (1980), this procedure indirectly labels one end of the 15 kb fragment and any fragments produced by DNAase digestion in this region. DNAase-hypersensitive sites will then appear on the autoradiograph as discrete bands whose size corresponds to the distance of the hypersensitive site from the labeled end. As shown in Figure 2a, one minor and two major DNAase-hypersensitive sites can
Cell 602
be detected within the 15 kb Sst fragment (from embryonic nuclei). These sites are located 2.1, 3.5 and 4.6 kb 3’ to the transcribed region of Sgs4. No hypersensitive sites are detected adjacent to the gene or in the 4 kb 5’ to the gene. Similar experiments with nuclei from the Schneider 2 cell line (Schneider, 1972) have revealed the same three 3’ sites and no others (data not shown). The sites identified in this way result from the chromatin conformation and not from an inherent preference of DNAase for a particular base sequence. When the substrate for DNAase digestion is naked DNA rather than nuclei, no prominent bands can be detected within Sgs4 or on either side of it (Figure 2b). DNAase Hypersensitive Sites near Sgs4 in Salivary Glands To determine whether the pattern of sites changes when Sgs4 is expressed, we mass-isolated salivary glands from mid-third instar larvae. To be sure that the glands came from larvae of the appropriate stage, some glands were squashed, and their polytene chromosomes were examined for puffed loci. The intermolt puffs 3Cl l-l 2 and 68C, which make major components of the glue (Korge, 1977; Akam et al., 19781, were visible, whereas the 74EF and 75B puffs, early members of the larval ecdysone-induced series (Ashburner, 19721, were not. Petri et al. (1977) have reported that during mass isolation of Drosophila tissues the heat-shock response may be induced. However, the heat-shock puffs at 87A and 87C were not visible in the chromosomes of our mass-isolated glands. Thus we conclude that the mass-isolated salivary glands are suitable for studies of Sgs4 chromatin structure. Nuclei from these glands were then digested briefly with DNAase I and analyzed as described above. The restriction enzyme used in the secondary digestion was Sal I, which produces a 19 kb restriction fragment whose left end is 2.45 kb 5’ to Sgs4 (Figure 1). When this is probed from the left end, a complex series of XdDml554 --
DNAase l-hypersensitive sites can be detected (Figure 3). The most prominent site is located about 400 bp 5’ to the Sgs4 cap site. Adjacent to this and extending approximately to -300 is a region that in this sample appears uniformly sensitive to DNAase I. The profile of this region is variable, appearing on some samples as a more defined peak at -330 (data not shown). Somewhat less sensitive DNAase I sites appear at -480, -70 and +30 (Figures 3 and 4). Near the top of the gel, the same 3’-hypersensitive sites seen in embryos can be observed. Because this experiment measured from the Sal I site 2-2.5 kb away, it located the hypersensitive sites with a resolution of +30-50 bp. We have been able to locate the most prominent site more accurately by following the DNAase treatment with digestion by Barn HI and Eco RI. As shown in Figure 1, this produces a fragment of 800 bp whose right end coincides with the right end of the gene (M. Muskavitch and D. Hogness, submitted). We then determined the position of the most prominent hypersensitive site in this interval by using as a probe either the Xho I-Eco RI fragment from the right end or the Barn HI-Xho I fragment from the left end. Both the Eco RI-Xho I fragment and the Eco RI-Sau 3A fragment were used as size standards, since they flank the DNAase site and their lengths have been determined by DNA sequencing (M. Muskavitch and D. Hogness, submitted). A densitometer tracing of one lane from this gel is
+10 F
I-
0
pDmRl5
,'
--_
,'
2
a
-5 I
1 kb
-.._
Figure 2. DNAase-Hypersensitive Chromatin and in Purified DNA 200t’p
Figure
1. Partial
Restriction
Map of the Sgs4 Region
(4) Transcribed region of Sgs4. Brackets above the map show the extent of genomic DNA cloned in hdDml554and pDmRl.5. For clarity only those restriction sites used in the present analysis are shown. More complete maps can be found in Muskavitch and Hogness (1980: submitted) and in McGinnis et al. (1980).
Sites
near
Sgs4
in Embryonic
(a) Embryonic nuclei were isolated and treated for 3 min with 0 or 15.6 units of DNAase I per milliliter. The DNA was then extracted, digested to completion with Sst I and analyzed by Southern blotting (Southern, 1975). with the use of the 1 .l kb Barn fragment isolated from AdDml554 d) as a probe. (b) Purified DNA rather than nuclei was treated for 75 set with 0.1 or 0.2 units of DNAase I per milliliter, digested with Sst I and analyzed as in (a). Thin lines connect corresponding regions of the diagram and the two gels. (h) DNAasehypersensitive sites.
Interacting 603
DNAase
l-Hypersensitive
ORE -
Sites
ORE+
HAS +
BER+ _ il
Figure 4. Summary of the Hypersensitive Sites Upstream from Sgs4 and Their Relation to the Deletions in the Oriental and BER 1 Strains Only the 5’ end of Sgs4 is shown. (9) Hypersensitive mentioned in the text, the site at -330 sometimes appears region (bracket). Solid bars below line: the two deletions.
sites. As as a broad
n
Figure 5. The Major DNAase bp Upstream from Sgs4
Figure 3. DNAase-Hypersensitive Mutant Salivary Gland Chromatin
Sites near Sgs4
in Wild-type
and
Salivary gland nuclei were isolated and treated for 3 min with DNAase I (0. 7.8, 7.8 or 5.2 U/ml for the four samples shown). After DNA extraction, the samples were digested with Sal I and analyzed by Southern blotting. The probe was the 0.31 kb Barn HI fragment isolated from pDmRl.5 (Figure 1). (+ and -) Treated or untreated with DNAase I. (p Position corresponding to the transcribed region of Sgs4. Locations of DNAase l-generated fragments 5’ to Sgs4 in the ORE+ and HAS+ lanes are indicated between the gel tracks. The bands near the top of the gel correspond to the 3’ hypersensitive sites. Their locations vary among the three strains as a result of differences in the length of the coding sequence (M. Muskavitch and D. Hogness. submitted) and of the 3’ flanking sequences.
shown in Figure 5. It locates the prominent -405 +5 bp from the Eco RI site.
site at
DNAase Hypersensitive Sites in Sgs4 Mutants We reasoned that the possible role of the Y-hypersensitive sites in Sgs4 expression might be clarified by analysis of SgsGdefective mutants, two groups of which have recently been characterized by Muskavitch and Hogness (1980; submitted). Strains in the Oriental group accumulate l-3% as much Sgs4 RNA as the Sgs4+ standard strain, Oregon R, and are missing 52 bp between -307 and -358 (M. Muskavitch and D. Hogness, submitted). The BER 1 strain does not produce detectable amounts of Sgs4 RNA and has a deletion of 103 bp between -394 and -496 (M. Muskavitch and D. Hogness, submitted). We have determined the pattern of DNAase-hypersen-
l-Hypersensitive
Site Is Located
405
DNA from salivary gland nuclei that had been treated with 7.8 units DNAase I/ml was digested with Barn HI and Eco RI and analyzed by Southern blotting with use of the 391 bp Xho I-Eco RI fragment that lies just 5’ to Sgs4 as the probe (see diagram above and Figure 1). The resulting autoradiograph was traced on a Joyce Loebl microdensitometer, and the position of the hypersensitive site was compared with standards from pBR322 and from pDmR1 S. a pBR322 derivative that contains the 1.5 kb Eco RI fragment that adjoins Sgs4 on the 5’ side. The two Drosophila fragments that flank the hypersensitive site are Xho I-Eco RI (391 bp) and Sau SA-(Eco RI (443 bp).
sitive sites near Sgs4 in salivary gland nuclei from an Oriental strain called Hikone AS and in BER 1. This pattern is distinctly altered in both cases. In Hikone AS, most of the 5’ sites are the same as those of Oregon R, except that the region of hypersensitivity extending from -400 to -300 is truncated at about -350, corresponding to the location of the deletion (Figure 3). Because the deletion is located between the labeled end of the restriction fragment and the two hypersensitive sites near the end of the gene (-70 and +30), these two appear to be displaced by about 50 bp in the 5’ direction. Their position relative to the gene, however, remains the same. Therefore, while the deletion removes a region that normally becomes hypersensitive, it does not affect the pattern of sensitivity in adjacent regions. There may, however, be an alteration in the relative frequencies with which the remaining sites are cut. The -70 and +30 sites appear to be underrepresented in the Hikone AS chromatin. The pattern is more drastically altered in BER 1.
Cell 604
Despite the fact that only the sequences corresponding to the two most distal sites are missing, no hypersensitive sites can be detected in BER 1 salivary gland nuclei in the region 5’ to Sgs4 (Figure 3). Thus the missing sequences appear to be required for downstream alterations in chromatin structure, as well as for expression of the gene. Discussion These results show that when Sgs4 becomes active in larval salivary glands, the chromatin structure 5’ to the gene is extensively altered so that five regions become accessible to DNAase I. In contrast, by the criterion of DNAase I hypersensitivity, the region 3’ to the gene is unaltered. A complex of hypersensitive sites has also been described in the region 5’ to several other active genes, including the Drosophila heat-shock genes (Wu, 1980; Keene et al., 1981), the rat preproinsulin gene (Wu and Gilbert, 1981) and the genes of polyoma virus (Herbomel et al., 1981). The availability of Sgs4 mutants has enabled us to learn something about the involvement of some parts of the complex in Sgs4 expression. In Hikone AS, the region of hypersensitivity corresponding to the DNA deletion is simply missing; the remainder of the hypersensitive sites are present and located over the same regions of DNA as in Oregon R. This result suggests that the deleted hypersensitive region is required for normal Sgs4 expression-without it, accumulation of Sgs4 message is reduced 50fold (M. Muskavitch and D. Hogness, submitted). This result also rules out a possible explanation for the formation of the hypersensitive sites. It might have been that sites appear at a defined distance from an active gene, regardless of the sequence located there. On such a model, the location of the sites might be dictated by steric or topological constraints on chromosome folding or supercoiling as the gene became accessible for transcription. Our results for Sgs4 are inconsistent with such a model, since the sites at -405 and -480 form over their normal sequences in Hikone AS, even though those sequences are 52 bp closer to the gene than they are in Oregon Ft. The second type of deletion mutant, BER 1, makes no detectable Sgs4 RNA and is missing a region of DNA that normally contains two hypersensitive sites, -405 and -480. In BER 1, not only are these sites missing, but also the other three 5’ sites are missing. This result implies that there are interactions among the various chromatin regions 5’ to Sgs4 and that they are hierarchical. Appearance of the three Sgs4 proximal sites is dependent on the sequences missing in BER 1, perhaps on the change in conformation which we detect as DNAase hypersensitivity. Although additional mutants will be required to distinguish between the -405 and -480 sites, we favor the idea
that the very prominent site at -405 is the one on which the remainder of the region is dependent. What kind of sequences correspond to the hypersensitive sites? There are many clusters of A-T-rich sequence 5’ to Sgs4 (M. Muskavitch and D. Hogness, submitted), and to the degree that we have localized the hypersensitive sites, all might be located in such clusters. However, we can rule out base composition as the sole criterion for hypersensitivity. There are many additional A-T-rich regions 5’ to Sgs4, most notably those between -100 and -300, which are flanked by hypersensitive sites but are not themselves sensitive. Several of the hypersensitive sites are located at or near specific sequences that may be important for gene expression. Two of the hypersensitive sites (- 70 and +30) are located near the beginning of the transcribed region. From comparison of many eucaryotic genes, three kinds of conserved sequences have been detected in this region. The sequence at the point of transcription initiation and attachment of the cap structure usually consists of an A residue surrounded by pyrimidines (Breathnach and Chambon, 1981). The Goldberg-Hogness or TATAA box approximately 23-30 bp 5’ to the cap site is very highly conserved and has been implicated in accurate transcription initiation (Goldberg, 1979; Grosschedl and Birnsteil, 1980; Benoist and Chambon, 1981; Grosveld et al., 1981; Mathis and Chambon, 1981). Benoist et al. (1980) have identified a third conserved sequence, GGPyCAATCT, often located 68-80 bp 5’ to eucaryotic genes. The region around this site has been shown to be essential for transcription of the herpes virus thymidine kinase gene (McKnight et al., 1981). Sgs4 has examples of all three of these sequences (M. Muskavitch and D. Hogness, submitted). Because of uncertainty regarding its exact location, the hypersensitive site that we have placed at -70 might correspond to either the GGPyCAATCT sequence or to TATAA. Similarly, the +30 hypersensitive site might correspond to the cap site. The three remaining hypersensitive sites are located 300-500 bp from the gene, in a region that has not been studied for many genes. However, there is some precedent for regulatory effects of sequences relatively far from the genes they control. Transcription in vivo of SV40 early genes requires sequences within the 72 bp direct repeats that are located 122-266 bp upstream (Benoist and Chambon, 1981; Gruss et al., 1981). In addition, transcription of the sea urchin H2A gene can be modulated 60 to 100 fold by loss or inversion of a segment normally located between -184 and -524 (Grosschedl and Birnsteil, 1980). The three Sgs4-hypersensitive sites are located over potentially interesting sequences. The -480 site corresponds to a threefold direct repeat and the -330 site to a partially selfcomplementary inverted repeat
interacting 605
DNAase
Table 1. Sequences
I-Hypersensitive
Located
Sites
5’ to Several
Drosophila
Genes Are Similar to the Sgs4 Sequence
Gene
Sequences
Distance Gene
sgs4
AAACTAAAGCTGGT
396 323/l
68C group
ll/lll
AAA.
68C group
IV
TAAAGCGCCT
References M. Muskavitch 57
M. Garfinkel. ,‘
AATATAAAGCATAT
420
hsp70
87A
AAA.
235
lngolia
hsp70
87C
AAAATAAAGCGAAT
235
Torok
copia
TAAAGAATAT
AATATAAAG.
TAGT
at -405
to and D. Hogness.
submitted
R. Pruitt and E. Meyerowitz,
et al. (1980); and Karch
Torok
and Karch
personal
communication
(1980)
(1980)
Rubin et al. (1980)
Five prominent RNAs have been identified in larval D. melanogaster salivary glands during the intermolt period (Wolfner. 1980). One of these is encoded by Sgs4 (McGinnis et al., 1980; Muskavitch and Hogness, 1980). Three others, called groups II. Ill and IV. are encoded by a cluster of genes located at 68C on the polytene chromosome map (M. Meyerowitz and D. Hogness. submitted). Two of these genes, II and Ill. are divergently transcribed, and the second sequence shown above is located between them. The third sequence is located 5’ to the group IV gene. Copies of the major D. melanogaster heat-shock gene, hsp70. are located at both 87A and 87C (Ashburner and Banner. 1979). Because the genes are slightly divergent at the two loci (Torok and Karch. 1980). one from each has been included. The copia sequence is located within the terminal direct repeat between positions 45 and 58 (Rubin et al., 1980).
(M. Muskavitch and D. Hogness, submitted). Perhaps the most interesting site is that centered at -405. It is the most sensitive to DNAase cleavage, and as discussed above, may be required for formation of the other sites and for transcription of the gene. Between -396 and -409 there is a 14 bp sequence that shares homology with sequences 5’ to the glue protein genes at 68C on the third chromosome and with sequences 5’ to copies of the heat-shock gene hsp70 located at bands 87A and 87C (Table 1). There is also a very similar sequence in the terminal direct repeat of the transposable element, copia. Whether in these other cases this sequence is involved in DNAase I hypersensitivity or is required for gene expression is not yet known. However, it seems unlikely to have occurred 5’ to all these genes by chance. In a random sequence, the invariant part of the sequence would be expected about once every 16 kb. In addition, a related sequence, GNNAAGNANNNAT, is found in the 72-nucleotide repeats of SV40 and the long terminal repeats of several mammalian retroviruses (S. K. Beckendorf, unpublished). Both the SV40 repeats and the retroviral long terminal repeats show DNAase l-hypersensitive sites when the associated genes are active (C. Cremisi and H. Weintraub, personal communication; Groudine et al., 1981). The SV40-hypersensitive site has been located within 15 bp of the conserved sequence. In conclusion, examination of the chromatin surrounding Sgs4 has revealed eight hypersensitive sites, five of which are tissue-specific. At least three of the five depend for their appearance on a region containing the most prominent site, which is located about 405 bp 5’ to the gene. This region also appears to be necessary for Sgs4 expression. Sequences related to that of the prominent hypersensitive site are found upstream from several other genes, suggesting that they are similarly dependent on DNAase Ihypersensitive regions.
Experimental
Procedures
Strains of Drosophila The Oregon R wild-type strain of D. melanogaster (obtained from W. Petri, Boston College) was used as the standard for DNA sequence arrangement near Sgs4. The two Sgs4 mutant strains used are Hikone AS (HAS; Bowling Green University) and Berkeley 1 (BER 1, originally identified as a laboratory stock that made no Sgs4 protein). The X chromosomes of these stocks were made isogenic before the experiments reported here were conducted. Collection of Embryos Embryos 6-18 hr old were obtained from a laboratory population of D. melanogaster (Oregon R) maintained at 25’C. as described by Elgin and Miller(l978). Embryos were washed extensively with water, dechorionated by stirring in 50% Clorox for 1 min and washed with distilled water, 70% ethanol and more distilled water. They were then quick-frozen at -8O’C until used. Salivary Gland Isolation Salivary glands were isolated from mid-third instar by a modification of the methods of Zweidler and Cohen (1971) and Boyd (1978). Briefly, larvae are reared en masse in disposable plastic dishes (Elgin and Miller, 1978) and isolated in late third instar from the walls of the dishes. The larvae are thoroughly washed to remove yeast and food particles and then suspended in organ medium: 25 mM disodium glycerophosphate; 10 mM KH,POI; 30 mM KCI; 10 mM MgC12. 3 mM CaCb and 162 mM sucrose (Cohen and Gotchel. 1971). All subsequent steps are carried out at 4’C in organ medium. The larvae are then disrupted by passage between two rotating stainless steel rollers that are spaced 0.18 mm apart. Large debris such as empty carcasses is removed by passage through Nitex screen (1 mm* pore size). Salivary glands are then purified by repeated settlings at unit gravity, centrifugal agitation (Zweidler and Cohen, 1971) and sedimentation through a 4 X 80 cm column containing a 5-10% gradient of Ficoll in organ medium. Most contaminating tissue remaining after these steps (mainly gut and Malpighian tubules) is removed through a drawn out Pasteur pipet attached to an aspirator. This procedure results in 90-95% pure salivary glands with a yield of 0.5-l ml per 200 g larvae. Isolation of Embryonic Nuclei Dechorionated embryos (5 g) were ground with a mortar and pestle at -70°C. The powdered embryos were then stirred into 20 ml of buffer A plus 0.25 M sucrose at 0°C (buffer A is 60 mM KCI, 15 mM NaCI. 1 mM EDTA. 0.1 mM EGTA, 0.15 mM spermine, 0.5 mM spermidine, 15 mM Tris-HCI [pH 7.41, 0.5 mM dithiothreitol and 0.1 mM phenylmethylsulfonyl fluoride [PMSF]: Wu et al., 1979) and
Cell 606
homogenized in a Dounce homogenizer fitted with a B pestle (six to eight strokes). The homogenate was filtered through 100 pm pore size Nitex screen, and the filtrate was centrifuged for 1 min at 2000 rpm (650 x g) in a Sorvall HB4 rotor. After discarding the pellet, the supernatant was brought to 0.2% Nonidet-P40. vortexed vigorously and centrifuged for 10 min at 6000 rpm (5860 X g) in the same rotor to pellet crude nuclei. The pellet was resuspended in buffer A plus 0.25 M sucrose and layered onto a crude gradient prepared from buffer A* plus 0.85 M sucrose and buffer A’ plus 1.7 M sucrose. (Buffer A* is buffer A without EDTA and EGTA.) The gradient was centrifuged for 20 min at 10,500 rpm (18,000 X g). The supernatant was removed, and the purified nuclear pellet was resuspended in nuclear buffer (60 mM KCI. 15 mM NaCI, 0.05 mM Car&, 0.1 mM EDTA, 15 mM Tris-HCI [pH 7.41. 0.5 mM dithiothreitol and 0.25 M sucrose). This procedure yielded nuclei containing about 0.5 mg DNA from each gram of embryos. Isolation of Salivary Gland Nuclei Two milliliters of purified salivary glands were suspended in 10 ml buffer A plus 0.25 M sucrose in a Dounce homogenizer and homogenized with four to eight strokes of an A pestle. Nonidet-P40 (10%; 250 Al) was added, and the suspension was vortexed until almost all cells had lysed, but nuclei were still intact. The suspension was strained through 110 Am Nitex and then centrifuged for 2 min at 1000 rpm in the HB4 rotor (164 X g). The crude nuclear pellet was then purified on the same sucrose gradient described for the embryonic nuclei. The resulting purified nuclei were resuspended in nuclear buffer. One milliliter of settled salivary glands yielded nuclei containing 100-l 50 Ag DNA. DNAase I Digestion DNAase I (DPFF grade) was purchased from Worthington. Just prior to use it was diluted in 60 mM KCI. 15 mM NaCI. 100 mM MgCI?, 0.1 mM CaC12. 15 mM Tris-HCI (pH 7.4) and 0.5 mM dithiothreitol. Digestions of intact nuclei were initiated by adding 25 Al of the appropriate DNAase I dilution to 475 pl of nuclear suspension at 25°C. After 3 min the digestion was stopped by adding 20 pl of 0.5 M EDTA. DNA Extraction Nuclei were lysed by addition of SDS to 0.5% and treated overnight at 37°C with proteinase K (E. Merck, Beckman) at 70 pg/ml. Each sample was extracted once with chloroform-isoamyl alcohol (24:1, v/v); once with equal volumes of chloroform-isoamyl alcohol and phenol: and once with ether. One volume of water was added, and RNA was removed by treatment with RNAase A and RNAase Tl (heattreated at 8O’C for 30 min to inactivate DNAases; final concentrations were 50 pgs/ml for RNAase A and 37 U/ml for RNAase Tl) at 30°C for 2 hr for 15 min. RNAases were removed by proteinase K treatment for 3 hr in 0.5% SDS at 37°C. and the protease was removed by a repetition of the organic extractions. The final aqueous phase was made 0.2 M in sodium acetate, and DNA was precipitated overnight at -20°C in 2.5 volumes ethanol. Preparation of Nick-Translated Probes Specific restriction fragments containing DNA from the Sgs4 region were isolated from the A derivative hdDm1554 (Muskavitch and Hogness. 1980) or the pBR322 derivative pDmRl.5, which was constructed by inserting the 1.5 kb Eco RI fragment located immediately 5’ to Sgs4 into the Eco RI site of pBR322. The extent of genomic DNA included in these clones is indicated in Figure 1. Afler appropriate restriction, the cloned DNAs were subjected to electrophoresis on agarose gels and visualized by ethidium-bromide staining. The desired fragment was excised from the gel and recovered by the glass fiber filter method of Davis et al. (1980). The fragments were then nick-translated according to the procedure of Maniatis et al. (1975). Southern Hybridizations Restriction endonucleases were obtained from New England BioLabs or Bethesda Research Laboratories and used according to the sup-
plier’s directions. Five micrograms of genomic DNA was digested per slot and fractionated on horizontal 0.556-l .O% agarose gels. Electrophoresis buffer was the Tris-borate-EDTA buffer described by Peacock and Dingman (1968). DNA fragments were transferred to diazotized paper, hybridized to nick-translated probe and washed by the method of Wahl et al. (1979). Autoradiography was performed at -8O’C by using a Du Pont Cronex Lightning Plus intensifying screen and Kodak XR-5 film. Before a previously probed DNA paper was reused, the old labeled probe was washed off by gently shaking the paper in 0.4 M NaOH for 15 min at room temperature and then rinsing thoroughly with distilled water. Acknowledgments We would like to thank Marc Muskavitch and David Hogness for continual communication of their results on Sgs4 organization and for providing us with several Sgs4 cloned sequences. We are grateful to Carl Wu and Sarah Elgin for instruction in DNAase digestion of chromatin; to Lawrence McGahey for preparation of nitrobenzyloxymethyl pyridinum chloride: and to Andrew Cockburn, John Farrell, Jr., Martha Fedor and William McGinnis for critically reading the manuscript. This research was supported in part by a Basil O’Connor Starter Research Grant from the March of Dimes Birth Defects Foundation and by a grant from the National Institute of Child Health and Human Development. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received
November
13. 1981;
Released
March
5, 1981
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Note Added
in Proof
The article referred to as “M. Muskavitch and D. Hogness, is now in press for the July 1980 issue of Cell.
submitted”